Johnson & Johnson (Australia)

BACKGROUND: MicroRNAs (MiRNAs) are short non-coding RNAs that control protein expression through various mechanisms. Their altered expression has been shown to be associated with various cancers. The aim of this study was to profile miRNA expression in colorectal cancer (CRC) and to analyze the function of specific miRNAs in CRC cells. MirVana miRNA Bioarrays were used to determine the miRNA expression profile in eight CRC cell line models, 45 human CRC samples of different stages, and four matched normal colon tissue samples. SW620 CRC cells were stably transduced with miR-143 or miR-145 expression vectors and analyzed in vitro for cell proliferation, cell differentiation and anchorage-independent growth. Signalling pathways associated with differentially expressed miRNAs were identified using a gene set enrichment analysis. RESULTS: The expression analysis of clinical CRC samples identified 37 miRNAs that were differentially expressed between CRC and normal tissue. Furthermore, several of these miRNAs were associated with CRC tumor progression including loss of miR-133a and gain of miR-224. We identified 11 common miRNAs that were differentially expressed between normal colon and CRC in both the cell line models and clinical samples. In vitro functional studies indicated that miR-143 and miR-145 appear to function in opposing manners to either inhibit or augment cell proliferation in a metastatic CRC model. The pathways targeted by miR-143 and miR-145 showed no significant overlap. Furthermore, gene expression analysis of metastatic versus non-metastatic isogenic cell lines indicated that miR-145 targets involved in cell cycle and neuregulin pathways were significantly down-regulated in the metastatic context. CONCLUSION: MiRNAs showing altered expression at different stages of CRC could be targets for CRC therapies and be further developed as potential diagnostic and prognostic analytes. The identified biological processes and signalling pathways collectively targeted by co-expressed miRNAs in CRC provide a basis for understanding the functional role of miRNAs in cancer.

Mammalian genome sequencing has identified numerous genes requiring functional annotation. The discovery that dsRNA can direct gene-specific silencing in both model organisms and mammalian cells through RNA interference (RNAi) has provided a platform for dissecting the function of independent genes. The generation of large-scale RNAi libraries targeting all predicted genes within mouse, rat and human cells, combined with the large number of cell-based assays, provides a unique opportunity to perform high-throughput genetics in these complex cell systems. Many different formats exist for the generation of genome-wide RNAi libraries for use in mammalian cells. Furthermore, the use of these libraries in either genetic screens or genetic selections allows for the identification of known and novel genes involved in complex cellular phenotypes and biological processes, some of which underpin human disease. In this review, we examine genome-wide RNAi libraries used in model organisms and mammalian cells and provide examples of how these information rich reagents can be used for determining gene function, discovering novel therapeutic targets and dissecting signalling pathways, cellular processes and complex phenotypes.

Heat shock protein 10 (Hsp10) and heat shock protein 60 (Hsp60) were originally described as essential mitochondrial proteins involved in protein folding. However, both proteins have also been shown to have a number of extracellular immunomodulatory activities. Here we show that purified recombinant human Hsp10 incubated with cells in vitro reduced lipopolysaccharide (LPS)-induced nuclear factor-κB activation and secretion of several inflammatory mediators from RAW264.7 cells, murine macrophages, and human peripheral blood mononuclear cells. Induction of tolerance as Hsp10 of with Hsp10 in of and and and of Hsp10 also in a murine to were to that Hsp10 and that Hsp10 with extracellular Heat shock protein 10 (Hsp10) and heat shock protein 60 (Hsp60) were originally described as essential mitochondrial proteins involved in protein folding. However, both proteins have also been shown to have a number of extracellular immunomodulatory activities. 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proteins involved in protein folding. 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vitro in were we to that Hsp10 a cells. that a described described that Hsp10 of to activation Hsp10 also to Hsp10 to to of activation of as However, we have been to Hsp10 with involved in Hsp10 of of extracellular with and Hsp10 and in that Hsp10 with in extracellular that Hsp10 and to and and that to of in with were to to involved in in of inflammatory been and Hsp10 of also to to and we have shown that Hsp10 also a Hsp10 in of to that of Hsp10 to that from from in of of Hsp10 to extracellular as that Hsp10 to and that Hsp10 and to secretion Hsp10 from of However, been shown to of been as a Hsp10 Hsp10 to and of in a murine of of Hsp10 to of inflammatory mediators that Hsp10 in to and of of recombinant

BACKGROUND: Short hairpin RNA (shRNA) encoded within an expression vector has proven an effective means of harnessing the RNA interference (RNAi) pathway in mammalian cells. A survey of the literature revealed that shRNA vector construction can be hindered by high mutation rates and the ensuing sequencing is often problematic. Current options for constructing shRNA vectors include the use of annealed complementary oligonucleotides (74 % of surveyed studies), a PCR approach using hairpin containing primers (22 %) and primer extension of hairpin templates (4 %). RESULTS: We considered primer extension the most attractive method in terms of cost. However, in initial experiments we encountered a mutation frequency of 50 % compared to a reported 20-40 % for other strategies. By modifying the technique to be an isothermal reaction using the DNA polymerase Phi29, we reduced the error rate to 10 %, making primer extension the most efficient and cost-effective approach tested. We also found that inclusion of a restriction site in the loop could be exploited for confirming construct integrity by automated sequencing, while maintaining intended gene suppression. CONCLUSION: In this study we detail simple improvements for constructing and sequencing shRNA that overcome current limitations. We also compare the advantages of our solutions against proposed alternatives. Our technical modifications will be of tangible benefit to researchers looking for a more efficient and reliable shRNA construction process.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) can play a major role in the management of acute pain in the peri-operative period. However, there are conflicting views on whether NSAIDs are associated with adverse renal effects. OBJECTIVES: The primary objective of this review was to determine the effects of NSAIDs on postoperative renal function in adults with normal preoperative renal function. SEARCH STRATEGY: Electronic searches for relevant randomised and quasi-randomised controlled trials in Cochrane Central Register of Controlled Trials, MEDLINE and EMBASE were performed. Attempts were also made to identify trials from citation lists of relevant trials, review articles and clinical practice guidelines. Handsearching of conference abstracts published in major anaesthetic journals was also performed. (Search date: 7 February 2003) SELECTION CRITERIA: The inclusion criteria were randomised or quasi-randomised comparisons of individual NSAIDs with either each other or placebo for treatment of postoperative pain, with relevant postoperative renal outcome measures, in adult surgical patients with normal renal function. DATA COLLECTION AND ANALYSIS: The data was extracted independently by two reviewers. The primary outcome measure was creatinine clearance within the first two days after surgery. Secondary outcome measures included serum creatinine, urine volume, urinary sodium level, urinary potassium level, fractional excretion of sodium, fractional excretion of potassium, need for dialysis and need for diuretic or dopamine treatment for renal insufficiency. Weighted mean differences for continuous outcomes and relative risk for dichotomous outcomes were estimated. MAIN RESULTS: Nineteen trials ( n = 1204) fulfilled the selection criteria for this review. NSAIDs reduced creatinine clearance by 16 ml/min (95%CI 5 to 28) and potassium output by 38 mmol/day (95%CI 19 to 56) on the first day after surgery compared to placebo. There was no significant difference in serum creatinine on the first day (0 umol/L, 95%CI -5 to 4) compared to placebo. No significant reduction in urine volume during the early postoperative period was found. There was no significant difference in serum creatinine in the early postoperative period between patients receiving diclofenace and ketorolac (or indomethacin). No cases of postoperative renal failure requiring dialysis were described. The trials were homogeneous for the primary outcome. REVIEWERS' CONCLUSIONS: NSAIDs caused a clinically unimportant transient reduction in renal function in the early postoperative period in patients with normal preoperative renal function. NSAIDs should not be withheld from adults with normal preoperative renal function because of concerns about postoperative renal impairment.

The 10-23 RNA cleaving DNAzyme has been shown to cleave any purine-pyrimidine (RY) junction under simulated physiological conditions. In this study, we systematically examine the DNAzymes relative activity against different RY combinations in order to determine the hierarchy of substrate core dinucleotide sequence susceptibility. The reactivity of each substrate dinucleotide compared in the same background sequence with the appropriately matched DNAzyme was found to follow the scheme AU = GU >> or = GC >> AC. The relatively poor activity of the DNAzyme against AC and GC containing substrates was found to be improved substantially by modifications to the binding domain which subtly weaken its interaction with the substrate core. The most effective modification resulting in rate enhancement of up to 200-fold, was achieved by substitution of deoxyguanine with deoxyinosine such that the base pair interaction with the RNA substrates core C is reduced from three hydrogen bonds to two. The increased cleavage activity generated by this modification could be important for application of the 10-23 DNAzyme particularly when the target site core is an AC dinucleotide.

The cellular uptake, intracellular distribution, and stability of 33-mer deoxyribozyme oligonucleotides (DNAzymes) were examined in several cell lines. PAGE analysis revealed that there was a weak association between the DNAzyme and DOTAP or Superfect transfection reagents at charge ratios that were minimally toxic to cultured cells. Cellular uptake was analyzed by cell fractionation of radiolabeled DNAzyme, by FACS, and by fluorescent microscopic analysis of FITC-labeled and TAMRA-labeled DNAzyme. Altering DNAzyme size and chemistry did not significantly affect uptake into cells. Inspection of paraformaldehyde-fixed cells by fluorescence microscopy revealed that DNAzyme was distributed primarily in punctate structures surrounding the nucleus and that substantial delivery to the nucleus was not observed up to 24 hours after initiation of transfection. Incubation in human serum or plasma demonstrated that a 3'-inversion modification greatly increased DNAzyme stability (t(1/2) approximately 22 hours) in comparison to the unmodified form (t(1/2) approximately 70 minute). The 3'-inversion-modified DNAzymes remained stable during cellular uptake, and catalytically active oligonucleotide could be extracted from the cells 24 hours posttransfection. In smooth muscle cell proliferation assay, the modified DNAzyme targeting the c-myc gene showed a much stronger inhibitory effect than did the unmodified version. The present study demonstrates that DNAzymes with a 3'-inversion are readily delivered into cultured cells and are functionally stable for several hours in serum and within cells.

PURPOSE: Myopia is a global public health issue; however, no information exists as to how potential myopia retardation strategies are being adopted globally. METHODS: A self-administrated, internet-based questionnaire was distributed in six languages, through professional bodies to eye care practitioners globally. The questions examined: awareness of increasing myopia prevalence, perceived efficacy and adoption of available strategies, and reasons for not adopting specific strategies. RESULTS: Of the 971 respondents, concern was higher (median 9/10) in Asia than in any other continent (7/10, p<0.001) and they considered themselves more active in implementing myopia control strategies (8/10) than Australasia and Europe (7/10), with North (4/10) and South America (5/10) being least proactive (p<0.001). Orthokeratology was perceived to be the most effective method of myopia control, followed by increased time outdoors and pharmaceutical approaches, with under-correction and single vision spectacles felt to be the least effective (p<0.05). Although significant intra-regional differences existed, overall most practitioners 67.5 (±37.8)% prescribed single vision spectacles or contact lenses as the primary mode of correction for myopic patients. The main justifications for their reluctance to prescribe alternatives to single vision refractive corrections were increased cost (35.6%), inadequate information (33.3%) and the unpredictability of outcomes (28.2%). CONCLUSIONS: Regardless of practitioners' awareness of the efficacy of myopia control techniques, the vast majority still prescribe single vision interventions to young myopes. In view of the increasing prevalence of myopia and existing evidence for interventions to slow myopia progression, clear guidelines for myopia management need to be established.

Only plants of the Papaver genus (poppies) are able to synthesize morphinan alkaloids, and cultivation of P. somniferum, opium poppy, remains critical for the production and supply of morphine, codeine and various semi-synthetic analgesics. Opium poppy was transformed with constitutively expressed cDNA of codeinone reductase (PsCor1.1), the penultimate step in morphine synthesis. Most transgenic lines showed significant increases in capsule alkaloid content in replicated glasshouse and field trials over 4 years. The morphinan alkaloid contents on a dry weight basis were between 15% and 30% greater than those in control high-yielding genotypes and control non-transgenic segregants. Transgenic leaves had approximately 10-fold greater levels of Cor transcript compared with non-transgenic controls. Two cycles of crossing of the best transgenic line into an elite high-morphine genotype resulted in significant increases in morphine and total alkaloids relative to the elite recurrent parent. No significant changes in alkaloid profiles or quantities were observed in leaf, roots, pollen and seed.

Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN or CD147), a member of the immunoglobulin family and a glycoprotein enriched on the surface of tumor cells, promotes invasion, metastasis, and growth and survival of malignant cells and confers resistance to some chemotherapeutic drugs. However, the molecular mechanisms underlying the actions of EMMPRIN are not fully understood. In this study we sought to determine whether EMMPRIN contributes to the malignant phenotype of breast cancer by inhibiting anoikis, a form of apoptosis induced by loss or alteration of cell-cell or cell-matrix anchorage, and to explore the signaling pathways involved. We found that in the absence of attachment, human breast carcinoma cells expressing high levels of EMMPRIN formed less compact aggregates with larger surface area and less fibronectin matrix assembly, had higher viability, and were resistant to anoikis. Knockdown of EMMPRIN expression by RNA interference (small interfering RNA or short hairpin RNA) sensitized cancer cells to anoikis, as demonstrated by activation of caspase-3, increased DNA fragmentation, and decreased cellular viability. Furthermore, we observed that the accumulation of Bim, a proapoptotic BH3-only protein, was reduced in EMMPRIN-expressing cells and that silencing of EMMPRIN expression elevated Bim protein levels and enhanced cellular sensitivity to anoikis. Treatment of cells with a MEK inhibitor (U0126) or proteasome inhibitor (epoxomicin) also up-regulated Bim accumulation and rendered cells more sensitive to anoikis. These results indicated that expression of EMMPRIN protects cancer cells from anoikis and that this effect is mediated at least in part by a MAP kinase-dependent reduction of Bim. Because anoikis deficiency is a key feature of neoplastic transformation and invasive growth of epithelial cancer cells, our study on the role of EMMPRIN in anoikis resistance and the mechanism involved underscores the potential of EMMPRIN expression as a prognostic marker and novel target for cancer therapy.

UNLABELLED: Retroviral vectors have been used in successful gene therapies. However, in some patients, insertional mutagenesis led to leukemia or myelodysplasia. Both the strong promoter/enhancer elements in the long terminal repeats (LTRs) of murine leukemia virus (MLV)-based vectors and the vector-specific integration site preferences played an important role in these adverse clinical events. MLV integration is known to prefer regions in or near transcription start sites (TSS). Recently, BET family proteins were shown to be the major cellular proteins responsible for targeting MLV integration. Although MLV integration sites are significantly enriched at TSS, only a small fraction of the MLV integration sites (<15%) occur in this region. To resolve this apparent discrepancy, we created a high-resolution genome-wide integration map of more than one million integration sites from CD34(+) hematopoietic stem cells transduced with a clinically relevant MLV-based vector. The integration sites form ∼60,000 tight clusters. These clusters comprise ∼1.9% of the genome. The vast majority (87%) of the integration sites are located within histone H3K4me1 islands, a hallmark of enhancers. The majority of these clusters also have H3K27ac histone modifications, which mark active enhancers. The enhancers of some oncogenes, including LMO2, are highly preferred targets for integration without in vivo selection. IMPORTANCE: We show that active enhancer regions are the major targets for MLV integration; this means that MLV preferentially integrates in regions that are favorable for viral gene expression in a variety of cell types. The results provide insights for MLV integration target site selection and also explain the high risk of insertional mutagenesis that is associated with gene therapy trials using MLV vectors.

Purpose The purpose of this paper is to investigate consumers' motivation to accumulate obsolete items and their reluctance to dispose of material possessions. Design/methodology/approach The role of attachment to material possession in the construction of consumer identity provides a conceptual framework for the research. A video‐ethnography with eight individuals, who classify themselves as functional hoarders, individuals who accumulate objects privately and are unable to dispose without clear conscious motivation or control, constitute the primary data for this paper. Findings In investigating the underlying reasons for accumulating objects and resisting dispossession, informants show evidence of being reflective consumers who perceive throwing away as a threat to memory, to security, and to historical and ecological preservation. First, this paper confirms current literature regarding the role of possessions as symbols of interpersonal ties with others and as a cue to past experiences. Second, the paper supports that possessions provide a sense of security to the owner. Finally, this paper reinforces that preserving material objects cultivate a vision for the future. Ultimately, informants' motivations to accumulate, to keep, and to not‐dispose of objects reflects a desire to reassemble the fragments of their temporal experience into a unique space where memories, present, and life projects join together. Originality/value The accompanying film gives an opportunity for audience members to personally evaluate hoarding practices and to draw their own conclusion on the dynamic nature of material attachment and consumer identity in terms of past experiences, present orientation, and responsibility for the future.

BACKGROUND: An anti-HIV-1 tat ribozyme, termed Rz2, has been shown to inhibit HIV-1 infection/replication and to decrease HIV-1-induced pathogenicity in T-lymphocyte cell lines and normal peripheral blood T-lymphocytes. We report here the results of a phase I gene transfer clinical trial using Rz2. METHODS: Apheresis was used to obtain a peripheral blood cell population from each of four HIV-negative donors. After enrichment for CD4+ T-lymphocytes, ex vivo expansion and genetic manipulation (approximately equal aliquots of the cells were transduced with the ribozyme-containing (RRz2) and the control (LNL6) retroviral vector), these cells were infused into the corresponding HIV-1-positive twin recipient. Marking was assessed over an initial 24-week period and in total over an approximate 4-year period. RESULTS: The gene transfer procedure was shown to be safe, and technically feasible. Both RRz2- and LNL6-gene-containing peripheral blood mononuclear cells (PBMC) were detected at all time points examined to 4 years. There was concomitant gene construct expression in the absence of the need for ex vivo peripheral blood cell stimulation and there was no evidence of immune elimination of the neoR T-lymphocytes nor of silencing of the Moloney murine leukemia virus long terminal repeat. CONCLUSIONS: The proof of principle results reported here demonstrate safety and feasibility of this type of gene transfer approach. While not specifically tested, T-lymphocytes containing an anti-HIV gene construct may impact on HIV-1 viral load and CD4+ T-lymphocyte count, potentially representing a new therapeutic modality for HIV-1 infection.

Early growth response factor-1 (Egr-1) controls the expression of a growing number of genes involved in the pathogenesis of atherosclerosis and postangioplasty restenosis. Egr-1 is activated by diverse proatherogenic stimuli. As such, this transcription factor represents a key molecular target in efforts to control vascular lesion formation in humans. In this study, we have generated DNAzymes targeting specific sequences in human EGR-1 mRNA. These molecules cleave in vitro transcribed EGR-1 mRNA efficiently at preselected sites, inhibit EGR-1 protein expression in human aortic smooth muscle cells, block serum-inducible cell proliferation, and abrogate cellular regrowth after mechanical injury in vitro. These DNAzymes also selectively inhibit EGR-1 expression and proliferation of porcine arterial smooth muscle cells and reduce intimal thickening after stenting pig coronary arteries in vivo. These findings demonstrate that endoluminally delivered DNAzymes targeting EGR-1 may serve as inhibitors of in-stent restenosis.

A small catalytic DNA molecule targeting c-myc RNA was found to be a potent inhibitor of smooth muscle cell (SMC) proliferation. The catalytic domain of this molecule was based on that previously derived by in vitro selection (Santoro, S. W., and Joyce, G. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4262–4266) and is known as the “10-23” general purpose RNA-cleaving deoxyribozyme. In addition to inhibiting SMC proliferation at low concentration, this molecule (targeting the translation initiation region of c-myc RNA) was found to efficiently cleave its full-length substrate in vitro and down-regulate c-myc gene expression in smooth muscle cells. The serum nuclease stability of this molecule was enhanced without substantial loss of kinetic efficiency by inclusion of a 3′-3′-internucleotide inversion at the 3′-terminal. The extent of SMC suppression was found to be influenced by the length of the substrate binding arms. This correlated to some extent with catalytic activity in both the short substrate under multiple turnover conditions and the full-length substrate under single turnover conditions, with the 9 + 9 base arm molecule producing the greatest activity. A small catalytic DNA molecule targeting c-myc RNA was found to be a potent inhibitor of smooth muscle cell (SMC) proliferation. The catalytic domain of this molecule was based on that previously derived by in vitro selection (Santoro, S. W., and Joyce, G. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4262–4266) and is known as the “10-23” general purpose RNA-cleaving deoxyribozyme. In addition to inhibiting SMC proliferation at low concentration, this molecule (targeting the translation initiation region of c-myc RNA) was found to efficiently cleave its full-length substrate in vitro and down-regulate c-myc gene expression in smooth muscle cells. The serum nuclease stability of this molecule was enhanced without substantial loss of kinetic efficiency by inclusion of a 3′-3′-internucleotide inversion at the 3′-terminal. The extent of SMC suppression was found to be influenced by the length of the substrate binding arms. This correlated to some extent with catalytic activity in both the short substrate under multiple turnover conditions and the full-length substrate under single turnover conditions, with the 9 + 9 base arm molecule producing the greatest activity. Antisense and ribozyme technologies are major tools in gene inactivation approaches for human gene therapy (2Stein C.A. Cheng Y.C. Science. 1993; 261: 1004-1012Crossref PubMed Scopus (1263) Google Scholar, 3Milligan J.F. Matteucci M.D. Martin J.C. J. Med. Chem. 1993; 36: 1923-1937Crossref PubMed Scopus (599) Google Scholar, 4Symonds R.H. Annu. Rev. Biochem. 1992; 61: 641-671Crossref PubMed Scopus (425) Google Scholar, 5Sun L.Q. Ely J.A. Gerlach W.L. Symonds G. Mol. Biotechnol. 1997; 7: 241-251Crossref PubMed Scopus (20) Google Scholar). However, these molecules (both as native nucleic acids and in modified forms) are highly susceptible to enzymatic hydrolysis and have potential for side effects in a cellular environment, thus limiting their pharmaceutical applications in a direct delivery mode. Recently, a new class of catalytic molecules made of single-stranded DNA (deoxyribozyme or DNA enzyme) was obtained through in vitro selection (1Santoro S.W. Joyce G.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4262-4266Crossref PubMed Scopus (1237) Google Scholar, 6Breaker R.R. Joyce G.F. Chem. Biol. 1995; 2: 600-655Abstract Full Text PDF Scopus (346) Google Scholar). One model denoted as the “10-23” deoxyribozyme was especially useful because of its ability to bind and cleave any single-stranded RNA at purine/pyrimidine junctions (1Santoro S.W. Joyce G.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4262-4266Crossref PubMed Scopus (1237) Google Scholar). This molecule is comprised of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of seven to eight deoxynucleotides each. Analysis has shown that this deoxyribozyme can efficiently cleave its substrate RNA with a catalytic rate of ∼0.1 min−1 andK M <1 nm under simulated physiological conditions in vitro. Given the catalytic efficiency, relative stability, and economy of production of DNA, an assessment of the therapeutic potential of this deoxyribozyme through cellular biochemistry is warranted. Restenosis is a major complication following angioplasty, occurring in 30–60% of patients (7Bittl J.A. N. Engl. J. Med. 1996; 335: 1290-1302Crossref PubMed Scopus (215) Google Scholar, 8Nobuyoshi M. Kimura T. Nosaka H. Mioka S. Ueno K. Yokoi H. Hamasaki N. Horiuchi H. Ohishi H. J. Am. Coll. Cardiol. 1988; 12: 616-623Crossref PubMed Scopus (738) Google Scholar). It is considered to be caused predominantly by vascular smooth muscle cell (SMC) 1The abbreviations used are: SMC, smooth muscle cell; ODN, oligodeoxynucleotides; bp, base pair; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylamino-niummethyl sulfate 1The abbreviations used are: SMC, smooth muscle cell; ODN, oligodeoxynucleotides; bp, base pair; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylamino-niummethyl sulfate proliferation after angioplasty. A variety of oncogenes, such as c-myc, c-fos, and c-myb, have been found to be involved in SMC proliferation and migration as well as deposition of extracellular matrix associated with post-vascular injury (9Banscota N. Taub R. Zellner K. King G. Mol. Endocrinol. 1989; 3: 1183-1190Crossref PubMed Scopus (112) Google Scholar, 10Gadeau A. Campna M. Dsgranges C. J. Cell. Physiol. 1991; 146: 356-361Crossref PubMed Scopus (36) Google Scholar, 11Gay C.G. Winkles J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 296-300Crossref PubMed Scopus (69) Google Scholar). These genes provide attractive targets for the prevention of restenosis by therapeutic agents that can specifically and locally suppress their expression in vivo. In this study we explore the use of synthetic deoxyribozymes targeted to c-myc RNA as potential therapy for restenosis. The anti-c-myc deoxyribozyme designed for this purpose was found to cleave efficiently its substrate RNA and mediate suppression of SMC proliferation with a concomitant reduction of c-MYC protein in transfected SMCs. We therefore demonstrate the potential of catalytic DNA as a new class of genetic therapeutic agents. All the oligonucleotides were made by Oligos Etc. (Wilsonville, OR) and purified by gel electrophoresis for in vitro cleavage studies and by high pressure liquid chromatography for cell-based assays. The efficacy of deoxyribozymes in vitro was determined by measuring the rate of RNA cleavage under multiple turnover conditions. For these experiments a range of substrate concentrations was used such that [S] ≥10-fold excess over [E] which was fixed at 200 pm. The deoxyribozyme oligonucleotide and a32P-labeled synthetic RNA substrate were pre-equilibrated separately for 10 min at 37 °C in 50 mm Tris·HCl, pH 7.5, 10 mm MgCl2, and 0.01% SDS. At time 0 the reaction was initiated by mixing the deoxyribozyme and substrate together. The reaction progress was then followed by the analysis of aliquots taken sequentially at various time points and quenched in 90% formamide, 20 mm EDTA, and loading dye. The product fragments and unreacted substrate in these samples were resolved by electrophoresis on a 16% denaturing polyacrylamide gel. The extent of reaction at each time point was determined by densitometry of the gel image produced through a PhosphorImager (Molecular Dynamics). The values for k obs (derived from the slope of these time course experiments) were used to generate a line of best fit in a modified Eadie-Hofstee plot (k obs versus k obs/[S]). In this expression the values for K M andk cat are given by negative slope of the regression line and the y intercept, respectively. For cleavage of full-length c-myc RNA, substrate RNA (1.5 kilobase pairs) was transcribed from a pGEM7-Zf(+) vector in the presence of [α-32P]UTP with an RNA transcription kit (Promega). Cleavage was carried out at 37 °C in a 10-μl volume containing 10 mm MgCl2, 50 mmTris·HCl, pH 7.5, 10 nm substrate RNA, and 50 nm deoxyribozyme oligonucleotide. Reaction was stopped at 60 min by adding equal volume of formamide loading buffer with EDTA, and the mixtures were then analyzed on a 6% denaturing polyacrylamide gel. Rat smooth muscle cells (SV40LT-SMC, ATCC CRL 2018) were cultured at 33 °C with 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% calf serum and 200 μg/ml G418. In the proliferation assay, smooth muscle cells were plated at 25,000 cells per well in a 6-well cluster plate and allowed to attach overnight. The following day, the cells were washed twice with PBS and then grown in 0.25% calf serum/DMEM for a period of 4 days at 33 °C. After 4 days, the media were replaced with 10% calf serum/DMEM, and the deoxyribozyme oligonucleotides were added as triplicate samples. Three days later, the cells are trypsinized and counted by a Coulter counter. Briefly, 150 μm unlabeled deoxyribozyme oligonucleotide was incubated in 100 μl of 100% human serum at 37 °C, and duplicate samples of 5 μl were removed at time points of 0, 2, 8, 24, 48, and 72 h. Immediately upon sampling 295 μl of Tris/EDTA was added to the 5-μl aliquot, and phenol/chloroform extraction was performed. All the samples from each time point were end-labeled with [γ-32P]ATP and run directly on 16% polyacrylamide gels without further purification or precipitation thus showing all intact oligonucleotides and degradation products. SMCs were arrested in serum-free medium for 72 h before incubation in methionine (Met)-free medium containing 5% dialyzed fetal calf serum. After 1 h at 37 °C this medium was removed and replaced with Met-free medium containing 5% dialyzed fetal calf serum, 100 μCi/ml [35S]Met, and 10 μm deoxyribozyme oligonucleotides and was incubated for a further 2 h. The cell lysates were prepared using the protocol as described (12), and c-MYC proteins were detected using a c-MYC-specific antibody C-8 (Santa Cruz Biotechnology). For Northern blot, total RNA was extracted from the SMCs treated with 2 μm deoxyribozyme or control oligonucleotides for 4 h. RNA was blotted on a membrane and hybridized with either c-myc-specific probe or glyceraldehyde-3-phosphate dehydrogenase fragment. Suppression of c-myc gene expression has been shown to be a promising cytostatic strategy for the prevention of restenosis. The reduction of c-myc is thought to be effective in this condition by inhibiting cell cycle progression in the early stage of the disease (13Libby P. J. Vasc. Surg. 1992; 15: 916-917Abstract Full Text PDF PubMed Scopus (4) Google Scholar, 14Simons M. Edelman E.R. Rosenberg R.D. J. Clin. Invest. 1994; 93: 2351-2356Crossref PubMed Scopus (108) Google Scholar, 15Shi Y. Hutchinson H.G. Hall D.J. Zalewsky A. Circulation. 1993; 88: 1190-1195Crossref PubMed Scopus (96) Google Scholar). In this study an RNA-cleaving deoxyribozyme designed (using the 10-23 model, Fig.1 A) to target the c-myc translation initiation codon (Fig. 1 B) was used. This site has been found to be an effective target for oligodeoxynucleotide (ODN)-mediated suppression of c-myc(15Shi Y. Hutchinson H.G. Hall D.J. Zalewsky A. Circulation. 1993; 88: 1190-1195Crossref PubMed Scopus (96) Google Scholar). The start codon in general has also been shown to be an amenable target for oligonucleotide-based gene inactivation (16Field A.K. Antiviral Res. 1998; 37: 67-81Crossref PubMed Scopus (16) Google Scholar). Multiple turnover kinetics were used to examine the efficiency of deoxyribozyme-catalyzed cleavage of a short synthetic c-myc RNA sequence in vitro. Three modified deoxyribozymes and their unmodified controls with symmetrical 7-, 8-, and 9-base pair substrate binding arms (Fig.2) were incubated with an excess of the32P-labeled synthetic c-myc RNA. From the values for k obs the kinetic parametersK M and k cat were determined (Fig. 3 and TableI).Figure 3Analysis of multiple turnover kinetics. A contains a plot of deoxyribozyme cleavage progress (nm) for each substrate concentration. All reactions were performed with 200 pm deoxyribozyme and 2, 4, 8, 16, and 32 nm substrate RNA (as indicated). B is a modified Eadie-Hofstee plot of the values for k obsdetermined at each substrate concentration. A line of best fit through these data can be used to obtain K M (negative slope) and k cat (y intercept).View Large Image Figure ViewerDownload (PPT)Table IKinetics of c-myc cleaving deoxyribozymesDeoxyribozymeArm lengthModificationk catK Mk cat/K Mmin−1nmpm−1· min−1Rs-17 + 7None0.252310.8Rs-27 + 73′ inversion0.16503.2Rs-38 + 8None0.113.432Rs-48 + 83′ inversion0.24460Rs-59 + 9None0.0678.6Rs-69 + 93′ inversion0.26465Kinetics of c-myc RNA cleavage was analyzed for three different length deoxyribozymes (both modified and unmodified) all targeting the start codon. Reactions were performed under multiple turnover conditions with at least a 10-fold excess of substrate in the presence 10 mm MgCl2 and 50 mmTris·HCl, pH 7.5. Open table in a new tab Kinetics of c-myc RNA cleavage was analyzed for three different length deoxyribozymes (both modified and unmodified) all targeting the start codon. Reactions were performed under multiple turnover conditions with at least a 10-fold excess of substrate in the presence 10 mm MgCl2 and 50 mmTris·HCl, pH 7.5. The overall catalytic efficiency of each deoxyribozyme as measured byk cat/K M values displayed a significant amount of variability between the modified and unmodified species. In the short arm deoxyribozymes (7 + 7 bp) the inclusion of an inverted base modification produced a 3-fold decrease in thek cat/K M, i.e. a decrease in catalytic efficiency. In contrast to this negative effect on the cleavage activity, the relative efficiency of the long (9 + 9 bp) arm version was enhanced 10-fold by the presence of inverted base modification. The intermediate length (8 + 8 bp) binding arm deoxyribozyme was the least affected by modification, showing a 2-fold increase in the value ofk cat/K M. The effect of the 3′-inverted terminal base was therefore different depending on the length of the substrate binding arms. A full-length c-myc RNA was used to test further the deoxyribozyme cleavage of more biologically relevant sequence. Cleavage reactions were performed under single turnover conditions, with 10 nmlong substrate (c-myc mRNA) and 50 nmdeoxyribozyme in 10 mm MgCl2, pH 7.5, 37 °C. The results demonstrated that all the deoxyribozymes could effectively cleave c-myc mRNA to an extent of 20–50% (Fig.4). The deoxyribozymes with longer arms cleaved substrates more efficiently, and 3′-inverted base modification decreased cleavage efficiency of the 7/7 arm deoxyribozyme but increased cleavage efficiency of the 9/9 arm deoxyribozyme. Interestingly, there was no difference in deoxyribozyme cleavage under conditions of either preheating the deoxyribozymes together with the c-myc RNA or no preheating. This indicated that the selected target site within the c-myc mRNA was very accessible in terms of RNA secondary structure in vitro. An assay was developed for examining deoxyribozyme oligonucleotide stability in 100% human AB serum. The results showed that the deoxyribozyme modified by a 3′-3′ inversion at the 3′ end had substantially greater stability in human serum (t12 = 20 h) compared with the unmodified deoxyribozymes that exhibited a half-life of <2 h (Fig.5). Anti-c-myc deoxyribozyme activity was in vascular smooth muscle cells M. Edelman E.R. Rosenberg R.D. J. Clin. Invest. 1994; 93: 2351-2356Crossref PubMed Scopus (108) Google Scholar). After in SMCs were from the addition of 10% calf These cells were to deoxyribozyme or control 9/9 arm deoxyribozyme with an inverted catalytic oligonucleotides in a containing suppression of SMC proliferation was determined at 72 h by of cell deoxyribozyme was shown to a decrease in cell compared with the control at 10 μm The effective range of the molecule 9/9 arms with 3′-inverted base was analyzed further in a The results indicated that this deoxyribozyme could significant suppression of SMC at concentrations to 50 nm (Fig. This cell proliferation was by the of given by the with deoxyribozyme cells showing no This was in contrast to the cells and treated with the control which displayed a increase in of c-myc deoxyribozyme on of smooth muscle were on the SMCs with or without The of per cells was determined were on the SMCs with or without The of per cells was determined Open table in a new tab demonstrate the activity of the at the the relative expression of c-MYC protein in and SMCs was determined by of SMCs with the deoxyribozyme was found to the of c-MYC protein to the in the cells with the control had no effect on in SMCs. Northern further this showing a of reduction in the c-myc mRNA caused by the deoxyribozyme in SMCs (Fig. 7 The length of the 10-23 deoxyribozyme substrate binding arms for target RNA cleavage in vitro on the target sequence of three human RNA targeted in vitro (1Santoro S.W. Joyce G.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4262-4266Crossref PubMed Scopus (1237) Google was shown that the deoxyribozyme activity with arms in target was by their to arms R.R. Joyce G.F. Chem. Biol. 1995; 2: 600-655Abstract Full Text PDF Scopus (346) Google Scholar). the target was the of the and deoxyribozymes with arms demonstrated substantially greater activity. The values M for each of these deoxyribozymes was found to with the stability of the N. S. M. A. H. T. M. M. 1995; PubMed Scopus Google Scholar). In the of the we cleavage efficiency in the unmodified with arms. the and of the unmodified c-myc deoxyribozyme had overall efficiency to their values cat/K M. The kinetic of these three different length molecules was by the inclusion of a This modification was to these molecules as a strategy for their stability in the presence of activity in J.F. H. H. A. A. M. H. Antisense Res. 1992; 2: PubMed Scopus Google Scholar). The of this DNA modification on the kinetics of cleavage was in the short arm deoxyribozyme. This molecule was substantially in terms of its value for k cat/K to the unmodified This reduction in catalytic efficiency was and enhanced by the addition of two in the modified This indicated that the reduction of activity in the short deoxyribozyme was to some of by the which could be by the arm to 8 in catalytic efficiency was found by the arm of the modified deoxyribozyme further to 9 This was in contrast to the in the unmodified deoxyribozyme that demonstrated a in activity between the with and arms. These results demonstrated that 8 is the arm length for c-myc RNA cleavage under these conditions by the unmodified the catalytic cleavage activity in the deoxyribozymes modified with a be to increase the length of each to 9 The in catalytic efficiency in the unmodified deoxyribozyme with arms was a reduction in turnover rate as a value for k This was a of an increase in the of the for the that product This reduction of activity was in the DNA modified by terminal base as a of of the All anti-c-myc deoxyribozymes were found to SMC after serum The effective of these deoxyribozymes symmetrical arms and was at the with a 3′-3′-internucleotide This with compared to the to the site on the c-myc which demonstrated to suppression of SMC proliferation under conditions Y. Hutchinson H.G. Hall D.J. Zalewsky A. Circulation. 1993; 88: 1190-1195Crossref PubMed Scopus (96) Google Scholar). The activity of the deoxyribozyme was significant to a of 50 This range was that with the ODN, with a substantial deoxyribozyme effect at concentrations The reduction in SMC proliferation was further in the which showed cells in a of in the treated compared with the The of suppression by at this c-myc site and targets is The is to the of and activity associated with these The of agents used to target the start codon of the c-myc target has been to because of the presence of a Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). the c-myc that this also known as a have been shown to of cells by a to their effect Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). This effect on proliferation can be from the of deoxyribozyme activity for a of the deoxyribozyme any and was at low concentrations the activity of was compared with a control oligonucleotide that was for an catalytic This oligonucleotide had very effect in cells at high concentrations the arm sequence. In had activity given that be of a It is that this molecule is a substrate for because of the sequence derived from the catalytic is that the activity of was derived from some of activity directly to the structure of this a control oligonucleotide that is by a point a inversion could be The activity of the various anti-c-myc deoxyribozymes correlated well with their activity in as through multiple turnover kinetics and cleavage efficiency on the full-length This is by the deoxyribozyme which the greatest suppression of SMC proliferation but also had the greatest kinetic efficiency under multiple turnover conditions and the cleavage of the full-length c-myc RNA this deoxyribozyme was found to a reduction in c-MYC protein in SMCs. was the decrease in the activity in the unmodified arm deoxyribozyme compared with the 3′-3′ deoxyribozyme This was showing the value of the in the oligonucleotide from However, this also have been the difference in cleavage kinetics between these two deoxyribozymes at the This is to some extent by the difference in activity between the modified and unmodified deoxyribozymes with arm The difference in kinetic efficiency between these modified and unmodified deoxyribozymes was also as as that for the arm in terms of k This is the to that deoxyribozyme could the of a new class of therapy with potential over and The 10-23 deoxyribozyme has the general as agents and greater catalytic activity that of results that the nuclease stability of DNA can be enhanced substantially by the of the base without the kinetic efficiency of the This also that deoxyribozymes are effective in a at both the and cellular We that a deoxyribozyme can be a inhibitor of c-myc expression in SMCs. The results described in this further that c-myc gene a in the of SMC proliferation. This the for further studies the therapeutic of anti-c-myc deoxyribozymes in vascular restenosis. In with an effective of the potential of deoxyribozymes as therapeutic in restenosis be

Cationic liposomes bind with nucleic acids such as plasmids and oligodeoxynucleotides to form complexes known as lipoplexes. Although these lipoplexes have several advantages over other forms of nucleic acid transfer methods in cell culture and in-vivo, toxicity remains a problem, especially in-vivo. Nevertheless, these carriers have been used in clinical trials against cystic fibrosis and cancer and their usage is attributed mainly to their versatility, especially when it comes to the range of routes available for administration of nucleic-acid-based drugs in-vivo.

We demonstrate that both over-expression and suppression of the gene encoding the morphinan pathway enzyme salutaridinol 7-O-acetyltransferase (SalAT) in opium poppy affects the alkaloid products that accumulate. Over-expression of the gene in most of the transgenic events resulted in an increase in capsule morphine, codeine and thebaine on a dry-weight basis. The transgenic line with the highest alkaloid content had 41%, 37% and 42% greater total alkaloids than the control in three independent trials over 3 years. DNA-encoded hairpin RNA-mediated suppression of SalAT resulted in the novel accumulation of the alkaloid salutaridine at up to 23% of total alkaloid; this alkaloid is not detectable in the parental genotype. Salutaridine is not the substrate of SalAT but the substrate of the previous enzyme in the pathway, salutaridine reductase. RNA transcript analysis of 16 primary T0 transformants and their segregating T1 progeny revealed an average reduction in SalAT transcript to about 12% of the control. Reduction in SalAT transcript was evident in both leaves and latex. Reverse transcriptase PCR and high-performance liquid chromatography analyses confirmed cosegregation of the expressed transgene with the salutaridine accumulating phenotype.

BACKGROUND: DzyNA-PCR is a general strategy for the detection and quantification of specific genetic sequences associated with disease or the presence of foreign agents. The method allows homogeneous gene amplification coupled with signal detection in a single closed vessel. METHODS: The strategy involves in vitro amplification of genetic sequences using a DzyNA primer that harbors the complementary (antisense) sequence of a 10-23 DNAzyme. During amplification, amplicons are produced that contain active (sense) copies of DNAzymes that cleave a reporter substrate included in the reaction mixture. The accumulation of amplicons during PCR can be monitored in real time by changes in fluorescence produced by separation of fluoro/quencher dye molecules incorporated into opposite sides of a DNAzyme cleavage site within the reporter substrate. The DNAzyme and reporter substrate sequences can be generic and hence can be adapted for use with primer sets targeting various genes or transcripts. RESULTS: Experiments using K-ras plasmid as template demonstrated that DzyNA-PCR allows quantification of DNA over at least six orders of magnitude (r = 0.992). Studies with human genomic DNA demonstrated the ability to resolve as little as twofold differences in the amount of starting template. DzyNA-PCR allowed the detection of 10 or fewer copies of the target. The clinical utility of the assay was demonstrated using DzyNA-PCR to analyze DNA that was isolated from human serum. CONCLUSION: DzyNA-PCR is a simple, rapid, and sensitive technique for homogeneous amplification and quantification of nucleic acids in clinical specimens.

PURPOSE: Many chemotherapeutic drugs have an inherent lack of safety due to interindividual variability of hepatic cytochrome P450 (CYP) 3A4 drug metabolism. This reduction in CYP3A4 in cancer patients is possibly mediated by cytokines associated with tumor-derived inflammation. We sought to examine this link by using an explant sarcoma in a novel transgenic mouse model of human CYP3A4 regulation. EXPERIMENTAL DESIGN: Engelbreth-Holm-Swarm sarcoma cells were injected into the hindlimb of transgenic CYP3A4/lacZ mice. Hepatic expression of the human CYP3A4 transgene was analyzed by direct measurement of the reporter gene product, beta-galactosidase enzyme activity. Hepatic expression of murine Cyp3a was analyzed at the mRNA, protein, and function levels. The acute phase response was assessed by examining cytokines [interleukin-6 (IL-6) and tumor necrosis factor] in serum, liver, or tumor as well as hepatic expression of serum amyloid protein P. RESULTS: Engelbreth-Holm-Swarm sarcoma elicited an acute phase response that coincided with down-regulation of the human CYP3A4 transgene in the liver as well as the mouse orthologue Cyp3a11. The reduction of murine hepatic Cyp3a gene expression in tumor-bearing mice resulted in decreased Cyp3a protein expression and consequently a significant reduction in Cyp3a-mediated metabolism of midazolam. Circulating IL-6 was elevated and IL-6 protein was only detected in tumor tissue but not in hepatic tissue. CONCLUSIONS: The current study provides a mechanistic link between cancer-associated inflammation and impaired drug metabolism in vivo. Targeted therapy to reduce inflammation may provide improved clinical benefit for chemotherapy drugs metabolized by hepatic CYP3A4 by improving their pharmacokinetic profile.

The vasculature of a tumour provides the most effective route by which neoplastic cells may be reached and eradicated by drugs. The fact that a tumour's vasculature is relatively more permeable than healthy host tissue should enable selective delivery of drugs to tumour tissue. Such delivery is relevant to carrier-mediated delivery of genetic medicine to tumours. This review discusses the potential of delivering therapeutic oligonucleotides (ONs) to tumours using cationic liposomes and cyclodextrins (CyDs), and the major hindrances posed by the tumour itself on such delivery. Cationic liposomes are generally 100-200 nm in diameter, whereas CyDs typically span 1.5 nm across. Cationic liposomes have been used for the introduction of nucleic acids into mammalian cells for more than a decade. CyD molecules are routinely used as agents that engender cholesterol efflux from lipid-laden cells, thus having an efficacious potential in the management of atherosclerosis. A recent trend is to employ these oligosaccharide molecules for delivering nucleic acids in cells both in-vitro and in-vivo. Comparisons are made with other ON delivery agents, such as porphyrin derivatives (< 1 nm), branched chain dendrimers (approximately 10 nm), polyethylenimine polymers (approximately 10 nm), nanoparticles (20-1,000 nm) and microspheres (> 1 microm), in the context of delivery to solid tumours. A discourse on how the chemical and physical properties of these carriers may affect the uptake of ONs into cells, particularly in-vivo, forms a major basis of this review.